F11 receptor (f11r) antagonists as therapeutic agents

ABSTRACT

The present invention provides a compound including a peptidomimetic which interacts sterically with the binding site of a F11R molecule, the peptidomimetic including a peptidomimetic having the SEQ ID NO: 4D. The present invention also provides a method for treating a disorder comprising administering peptide 4D to a mammal.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. Ser. No. 13/156,883filed on Jun. 9, 2011, which is a Continuation-in-Part of U.S. Ser. No.12/358,352 filed on Jan. 23, 2009, now abandoned, which is aContinuation-in-Part of U.S. Ser. No. 12/141,635 filed on Jun. 18, 2008,now U.S. Pat. No. 7,829,663 issued on Nov. 9, 2010, which is adivisional of U.S. Ser. No. 11/173,037, filed on Jul. 1, 2005, nowabandoned, which was a Continuation-in-Part of PCT/US2003/39890, filedon Dec. 16, 2003, which claims the benefit of U.S. ProvisionalApplication No. 60/438,669 filed on Jan. 3, 2003, the entire contents ofwhich are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to protein and peptide chemistry, as wellas crystallography and organic chemistry. The present invention isdirected to a cell adhesion molecule (CAM) and fragments thereof, andmore particularly to a CAM designated as the F11 receptor (F11R), or apolypeptide fragment thereof. The present invention also relates toF11R-antagonists and methods for the prevention and treatment ofexcessive bleeding following a wound injury, inflammatory diseases ofthe nervous system, thrombosis, inflammatory thrombosis,atherothrombosis, atherosclerosis, angiogenesis, plaque formation,cancer, immunothrombocytopenia (ITP), heart attacks, stroke, disordersof platelet and endothelial cell dysfunctions and other disordersinvolving thrombus formation, and hypertension.

BACKGROUND OF THE INVENTION

The vasculature is recognized as a dynamic metabolic organ that existsunder normal physiological conditions in an intact, undisturbed state(Karsan, et al. In: Hematology: Basic Principles and Practice, 3rd Ed.Hoffman, et al. (eds) 2000; pp. 1770-82). Endothelial cells (EC), whichline the exposed (luminal) surface of blood vessels, are normally notthrombogenic. That is, healthy EC do not attract or bind circulatingplatelets (Cines, et al. Blood 1998, 91: 3527-61; May, et al. ThrombHaemost 1999, 82: 962-70). It is well known that the physiologicalfunction of the endothelium is to facilitate blood flow by providing ahighly thromboresistant surface to flowing blood that inhibits plateletadhesion and clotting (Cines, et al.). However, under inflammatoryconditions, the nonthrombotic surface of EC can be transformed to aprothrombotic surface following exposure to cytokines (May, et al.;Diquelou et al. Thromb Haemost 1995, 74: 778-83), resulting inprocoagulant activity and a predisposition to thrombosis (May, et al.;Dardik, et al. Br J. Haematol. 2000, 109:512-8; Andre, et al. Blood2000, 96:3322-8). Indeed, the adhesion, accumulation and recruitment ofnon-stimulated platelets on cytokine-stimulated EC have been reported,with studies implicating the Platelet Endothelial Cell AdhesionMolecule-1 (PECAM-1; Rosenblum, et al. Stroke 1995, 27:709-11); beta 1integrin (Bombeli et al. J Exp. Med. 1998, 187:329-39), von Willebrandfactor (Dardik, et al.; Andre, et al.), and tissue factor (Verheul, etal. Blood 2000, 96:4216-21) in these processes. Thus, under inflammatoryconditions, cytokines induce alterations in EC which result in theadhesion of non-stimulated platelets.

Recently, a novel adhesion protein of the immunoglobulin (Ig)superfamily has been described with properties indicating a potentialtriggering role in the pathogenesis of inflammatory thrombosis,atherosclerosis and other disorders involving thrombosis formation. Thisprotein was identified first on the surface of human platelets andcalled the F11 receptor (F11R; Kornecki, et al. J Biol Chem 1990, 265:10042-8; Naik, et al. Biochem J 1995, 311: 155-62), and then on thesurface of murine endothelial and epithelial cells and called JAM/JAM-A(Martin-Padura, et al. J. Cell Biol. 1998, 142:117-27.).

The human platelet F11 receptor (F11R) is a surface glycoprotein duplex(32 and 35 kD at core protein: 29 kDa) member of the immunoglobulinsuperfamily. The Fl1R was first discovered as the target of a potentstimulatory monoclonal antibody, M.Ab.F1l, that induces plateletsecretion followed by aggregation (Kornecki, et al.; Naik, et al.;Kornecki, et al. J Lab Clin. Med. 1988, 111:618-26; Wang et al. Biochem.J. 1995, 311: 401-6; Kornecki, et al. In: Leukocyte Typing V.Schlossman, et al. (eds.) Oxford University Press 1195: 1241-3; Sobocka,et al. Blood 1997, 90: 10, Supplement 1, Part 2, Nov. 15, 2996a.;Sobocka, Ph.D. Thesis, 1998: SUNY Downstate, Brooklyn, N.Y., PresentedJun. 10, 1998; published Sep. 15, 1998; Sobocka, et al. Blood 2000,95:2600-9; Babinska, et al. Thromb Haemost 2002, 87: 712-21). Signaltransduction mechanisms for platelet secretion and aggregation inducedby M.Ab.F11 following its initial binding to F11R include: crosslinkingof the F11R to the FcγRII (Naik, et al.), activation and translocationof specific PKC isozymes (Wang, et al.), phosphorylation of the F11Rthrough activation of PKC (Naik, et al.; Wang, et al.), phosphorylationof the F11R following induction of platelet aggregation by thephysiological agonists thrombin and collagen and by M.Ab.F11 itself(Sobocka, et al. 1997; Sobocka; Sobocka, et al. 2000; Babinska, et al.),and phosphorylation of myosin light chain and pleckstrin, leading toshape change and granular secretion respectively (Kornecki, et al.1990). Following secretion, this signal transduction pathway culminatesin the activation of latent fibrinogen receptors and plateletaggregation (Kornecki, et al. 1990). Partial amino acid sequencesrepresenting 30% of the length of purified F11R have been reported byKornecki in 1995 (Naik, et al.). Cloning of the full-length cDNA for theplatelet F11R has revealed that it is a cell adhesion molecule (CAM), amember of the immunoglobulin superfamily (Sobocka, et al. 1997; Sobocka;Sobocka, et al. 2000). As a CAM, the F11R participates in mechanismsunderlying adhesion of platelets, endothelial cells, and epithelialcells (Martin-Padura, et al.; Sobocka, et al. 2000).

The conclusion that, in addition to its role as a receptor that triggerssignal transduction leading to secretion, the F11R also serves as a CAMinvolved in platelet adhesion was supported by the high degree ofsequence similarity found between the human platelet F11R and anadhesion protein called Junctional Adhesion Molecule (JAM), a proteinfound in murine endothelial cells (Martin-Padura, et al. 1998).Comparison of the murine JAM sequence to the previously-reportedsequences of the human platelet F11R (Naik, et al.) revealed over 70%homology of JAM to the N-terminus (23 amino acids) of F11R and to twoenzyme-digested products of F11R. In addition, both the human plateletF11R core protein and the murine JAM protein were found to contain asingle transmembrane domain and two pairs of cysteine residues in theirextracellular domains that allow formation of intermolecular disulfidebridges forming characteristic Ig-like folds. It is now well establishedthat the protein referred to as JAM (Martin-Padura, et al, 1998; Ozaki,et al. J. Immunol. 1999, 163: 553-7; Williams, et al. Mol. Immunol.1999, 36: 1175-88; Liu, et al. J. Cell Science 2000, 113: 2363-74;Gupta, et al. IUBMB Life 2000, 50: 51-6; Naik, et al. J. Cell Science2001, 114: 539-47), is the murine ortholog of the human F11R (Kornecki,et al. 1990; Naik, et al 1995; Kornecki, et al. 1988; Wang, et al.;Kornecki, et al 1995; Sobocka, et al. 1997; Sobocka; Sobocka, et al.2000; Babinska, et al.). JAM was localized at intercellular junctions ofmouse endothelial and epithelial cells (Martin-Padura, et al.).Similarly, the platelet antibody M.Ab.F11 was found to recognize F11Rmolecules present at intercellular junctions of cultured human umbilicalvein endothelial cells (Sobocka, et al. XVIII ISTH Congress, July, 2001,Paris, France, Abs #P1902; Babinska et al., manuscript submitted, 2005).A recent study conducted by the inventors (Babinska, et al. 2002) hasdetermined that two domains of F11R are critical for the induction ofplatelet aggregation by M.Ab.F11 and the adhesion of platelets toM.Ab.F11. Heretofore, the role of F11R in physiological andpathophysiological processes involving the adhesion of platelets tocytokine-inflamed endothelial cells has remained unknown. The inventorshave now determined that the N-terminus of F11R and the first Ig fold ofF11R contain protein sequences which are critical for the adhesion ofplatelets to endothelial cells, and that the recombinant soluble F11Rprotein and F11R-peptides block approximately 60% of the force ofadhesion of platelets to cytokine-treated EC, demonstrating theinvolvement of the F11R protein in platelet-endothelial cellinteractions, which under pathological conditions, result in thrombosis,atherosclerosis and other disorders involving thrombosis formation.

SUMMARY OF THE INVENTION

The present invention provides the full length cDNA sequence of the F11receptor (F11R) (SEQ ID NO: 6) and the encoded F11R amino acid sequence(SEQ ID NO: 7). The present invention also provides F11R-antagonistsincluding antibodies directed to the F11R as antagonists, peptideantagonists and peptidomimetic drugs that inhibit the biological actionof the F11R protein.

The present invention further provides a therapeutic regimen or plan oftreatment to inhibit platelet aggregation and adhesion, comprisingadministering F11R antagonist(s) separately or together with one or moreadditional compounds that contribute to a more favorable therapeuticoutcome, e.g., treating and preventing the formation of atheroscleroticplaques, thrombosis, and the like. The regimen (i.e., a plan or courseof treatment) for decreasing platelet aggregation and/or adhesionbetween platelets and endothelial cells in a subject in need thereof,comprises, administering to the subject an amount of an F11 receptor(F11R) antagonist(s) suitable to inhibit platelet aggregation and/or theadhesion between the endothelial cell and the platelet.

The present invention is directed to methods and compositions fortreating F11R-mediated disorders such as thrombosis, atherosclerosis,plaque formation, heart attacks, inflammatory diseases of the nervoussystem, hypertension, stroke, angiogenesis, cancer, and all otherclinical disorders involving thrombus formation, as detailed above. Theinvention is also directed to methods for the treatment and preventionof excessive bleeding under physiological procedures, including theprevention of excessive bleeding following wound injury. The presentinvention provides specific compositions containing at least oneF11R-antagonist peptide which inhibits, suppresses or causes thecessation of at least one F11R-mediated biological activity in a mammal,and preferably humans. Another embodiment of the present invention isthe preparation of peptidomimetic drugs that have a structure thatmimics the active site of F11R and thus inhibit its biological action.An example of the relationship of the structure of such a drug to thestructure of the F11R protein is the relationship between the structureof morphine and the protein beta-endorphin.

Nucleic acid molecules coding for any of the above F11R-antagonistproteins, fragments and peptides of the present invention, expressionvectors which include any of such nucleic acid molecules, as well asrelated host cells containing such nucleotide sequences or vectors, arealso contemplated by the present invention.

Still another embodiment of the present invention is directed toantibodies raised against the F11R-antagonist proteins, F11R antibodyfragments, peptidomimetics and peptides of the present invention.

In accordance with the present invention, the F11R antagonists encompasscompounds that mimic F11R, which when present in sufficient amounts willoccupy one or more relevant molecular and/or cellular F11R binding sitesthat normally would be occupied by F11R in its role as a mediator ofplatelet aggregation and adhesion. These compounds include smallmolecule organic compounds, peptides, peptidomimetics, or polypeptides,that structurally resemble a portion of F11R and compete for F11Rbinding sites on homophilic or heterophilic F11R binding partners.

F11R antagonists also encompass compounds, peptides and polypeptidesthat bind directly to F11R thereby impairing its aggregative/adhesivefunction. This group encompasses small molecule organic compounds,ligands, polypeptides, antibodies or antibody fragments, recombinantengineered proteins and antibodies that specifically bind to a region onF11R thereby inhibiting its participation in platelet aggregation andadhesion.

In an even additional embodiment of the invention, the peptide orpolypeptide may comprise the extracellular domain of F11R or anaggregation/adhesion inhibiting portion thereof. Non-limiting examplesof such portions of the F11R extracellular domain are exemplified by SEQID NO: 1 or SEQ ID NO: 4. In general, more than one antagonist may beadministered. This may be especially desirable when the administeredantagonists have distinct modes of inhibition, such as: (a) oneantagonist is from Group A and one is from Group B; (b) each of theadministered antagonists is from Group A, but each mimics distinctportions of the F11R, and (c) each of the administered antagonists isfrom Group B, but each of the antagonist binds to a distinct portion ofthe F11R, thereby inhibiting the aggregating/adhesion functions of therespective portions of F11R.

With respect to embodiments encompassing antibody antagonists, theantibody or functional fragment thereof can be a chimeric antibody, ahumanized antibody or an autoantibody. Non-limiting examples of suchantibodies comprise M.Ab.F11, the Fab, Fab′, or F(ab′)₂.

Preferably, the antibodies of the present invention are raised againstthose F11R sequences and F11R-antagonist peptides whose sequencescoincide with the corresponding sequences of a mammalian F11R orJunctional Adhesion Molecule (JAM) proteins. The antibodies of thepresent invention can recognize, antagonize or neutralize the activityof F11R. Both polyclonal antibodies and monoclonal antibodies of variouschimeric combinations are contemplated by the present invention.Examples of such antibodies include M.Ab.F11.

An additional embodiment of the present invention encompasses a methodof inhibiting platelet aggregation or platelet adhesion to anendothelial cell in a subject in need thereof, comprising:

administering an antagonist of F11R that either binds to theextracellular portion of F11R, or mimics the extracellular domain ofF11R or portions thereof, in an amount sufficient to inhibit plateletaggregation or platelet adhesion to endothelial cells. In accordancewith the present invention, the F11R antagonist comprises a compound, apeptide, a peptidomimetic, or an antibody or functional fragment thereofthat inhibits F11R-mediated aggregation and adhesion. The antibody orfunctional fragment thereof can be a chimeric or fully humanizedantibody or an autoantibody. Further, the peptide can comprise theextracellular domain of F11R or an aggregation/adhesion-inhibitingportion or fragment thereof.

The present invention also provides a compound including apeptidomimetic which interacts sterically with the binding site of aF11R molecule, the peptidomimetic including a peptidomimetic having theSEQ ID NO: 4D. The present invention further provides a method fortreating a disorder comprising administering peptide 4D to a mammal.

These and other embodiments of the invention will be readily apparent tothose of ordinary skill in view of the disclosure herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 demonstrates that adding a suitable amount of purified solubleextracellular portion of F11R (“sF11R”) to platelet suspensions inhibitsplatelet aggregation in a concentration-dependent manner. Inhibition ofM.Ab.F11 (M.Ab.F11R; 2.45 μg/ml)-induced platelet aggregation by theaddition of sF11R (soluble F11R) was observed at two concentrations ofsF11R, 1 μg/ml (1A) and 4 μg/ml (1B), with complete, irreversibleplatelet aggregation observed at 4 μg/ml sF11R (1B). Control tracingshow the full-blown platelet aggregation that occurred in the absence ofsF11R. These results indicate that sF11R, and therefore native F11R aswell, comprise at least one peptide sequence that is sufficient toinhibit M.Ab.F11-induced platelet aggregation.

FIG. 2 illustrates the assay for the potentiation of ADP-inducedplatelet aggregation by sub-threshold, i.e., non-aggregating, amounts ofM.Ab.F11 and adenosine diphosphate (“ADP”). The platelets do notaggregate in the presence of sub-threshold (i.e., non-aggregating)concentrations of either ADP alone (0.5 μM) or M.Ab.F11 alone (0.3μg/ml) (see the flat recordings in FIG. 2A-B, respectively). However, incombination, the sub-threshold amounts of ADP and M.Ab.F11 induce fullblown aggregation recordings as shown in FIGS. 2A-B (tracings labeledM.Ab.F11 plus ADP). Further, the aggregation-inhibiting activity of twospecific F11R-derived peptides corresponding to SEQ ID NO: 1 and SEQ IDNO: 4 are exemplified in FIGS. 2C-D, respectively. As shown in FIG. 2D,experiments were carried-out in which the peptide of SEQ ID NO: 1 (50μM) was preincubated with platelets for about 30 sec prior to theaddition of M.Ab.F11 (0.3 μg/ml), followed by the addition of ADP (0.5μM). FIG. 2D demonstrates that Peptide 1 SEQ ID NO: 1 completelyinhibited platelet aggregation (see the flat tracing, “plus Peptide 1”).Similarly, experiments were carried-out (see FIG. 2C) in which theF11R-peptide of SEQ ID NO: 4 was preincubated with platelets for about42 sec prior to the addition of M.Ab.F11 (0.3 μg/ml) followed byaddition of ADP (0.5 μM). FIG. 2C demonstrates that Peptide 4 SEQ ID NO:4 completely inhibited aggregation (see the flat tracing labeled “plusPeptide 4”). The results of similar experiments using the peptides ofSEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 5 do not inhibit aggregationin the assay (Results not shown).

FIG. 3 shows inhibition by F11R peptides (SEQ ID NOS: 1 and 4) in aplatelet adhesion assay in which monoclonal antibody M.Ab.F11 wasimmobilized and the binding of platelets to immobilized M.Ab.F11 wasmeasured.

FIG. 4 illustrates the inhibitory effects of two concentrations of Fabfragments prepared from monoclonal antibody M.Ab.F11 in the plateletaggregation assay. The Fab fragments were added at concentrations of 0.1μg/ml (a low concentration) (panel a) or 1.0 μg/ml, (panel b), a 10-foldhigher concentration which completely inhibited platelet aggregation.

FIG. 5 illustrates Fab fragment inhibition of the adhesion of eithernon-activated platelets (FIG. 5A) or collagen-activated platelets (FIG.5B) to proinflammatory cytokine, TNF-α stimulated-endothelial (HAEC,human aortic endothelial cell) monolayers. FIG. 5A shows that Fabfragments of antibody M.Ab.F11 blocked 20% of the adhesion ofnon-activated platelets to the aortic cells, whereas FIG. 5Bdemonstrates that the Fab fragments were effective in blocking over 90%of the adhesion of collagen-activated platelets to the endothelial cellmonolayers.

FIG. 6 illustrates inhibition by the Fab fragment of M.Ab.F11 of theadhesion of non-activated platelets (FIG. 6A), or collagen-activatedplatelets (FIG. 6B) to HAEC monolayers treated with a proinflammatorycytokine combination comprised of both TNF-α plus INF-γ. As shown inFIG. 6A, a 60% inhibition of the adhesion of non-activated platelets tothe cytokine-treated HAEC monolayers could be achieved in the presenceof 100 μg Fab (BAR 2), and 80% inhibition of adhesion occurred in thepresence of 200 μg/ml Fab (BAR 3). Similarly, as shown in FIG. 6B, a 70%inhibition of the adhesion of collagen-activated platelets to theendothelial cells could be achieved in the presence of 100 μg Fab (BAR2), whereas a stronger inhibition (95% inhibition) of the plateletadhesion to HAEC could be achieved in the presence of 200 μg Fab (BAR3). (Values obtained in control experiments, “no Fab added”, were set at100%).

FIG. 7A illustrates the appearance of the untreated group of animals;

FIG. 7B illustrates the physical appearance of the treated group ofanimals;

FIG. 7C illustrates the appearance of the untreated group of animals;

FIG. 7D illustrates the physical appearance of the treated group ofanimals;

FIG. 7E illustrates the appearance of the untreated group of animals;

FIG. 7F illustrates the physical appearance of the treated group ofanimals;

FIG. 8A illustrates a comparison of plaque formation between anuntreated animal and a treated animal;

FIG. 8B illustrates a comparison of plaque formation between anuntreated animal and a treated animal;

FIG. 8C illustrates a comparison of plaque formation between anuntreated animal and a treated animal;

FIG. 9A illustrates a comparison of the aortae of between treated anduntreated animals;

FIG. 9B illustrates a comparison of the aortae of between treated anduntreated animals;

FIG. 9C illustrates a comparison of the aortae of between treated anduntreated animals;

FIG. 9D illustrates a comparison of the aortic lesions between treatedand untreated animals;

FIG. 10A illustrates a comparison between atherosclerotic plaques oftreated and untreated animals;

FIG. 10B illustrates a comparison between atherosclerotic lesions oftreated and untreated animals;

FIG. 10C illustrates a comparison between atherosclerotic lesions oftreated and untreated animals; and

FIG. 10D illustrates a comparison between atherosclerotic lesions oftreated and untreated animals.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to F11R-antagonists and particularlyantibody directed against F11R, the Fab, Fab′, F(ab′)₂ fragments of suchantibody, as well as single-chain anti-F11R antibodies. By“F11R-antagonist” is meant molecules that inhibit, suppress or cause thecessation of at least one F11R-mediated biological activity by, e.g.,interfering with, blocking or otherwise preventing or regulating theinteraction or binding of F11R to its target, e.g. F11R on another cell,or another protein that F11R binds to such as itself or other JAMs, tothe leukocyte function associated antigen-1 (LFA-1) (Ostermann et al.,2002, Nat. Immunol. 3, 151-158), the integrins GPIIb/IIIa and α_(v)β₃,as well as other binding proteins.

In accordance with the present invention, novel F11R-antagonist peptidesderived from or corresponding to the F11R have been isolated andsynthesized. These peptides possess F11R antagonistic propertiesincluding the ability to selectively bind to F11R and inhibitF11R-mediated biological activity which, for example, is associated withadhesion of platelets to endothelial cells in mammals. The peptides ofthe present invention preferably correspond to specific portions of thenative human F11 receptor and include variations thereof, and thereforeare non-immunogenic when administered to humans. The peptides of thepresent invention can effectively block collagen-induced plateletaggregation and secretion and thereby are efficacious in regard to,inter alia, the prevention of excessive bleeding following an injury,under physiological conditions. Moreover, under pathological conditions,the uncontrolled accumulation of platelets in the vasculature, on theluminal surface of the inflamed endothelium or at exposed collagen siteswithin the injured vasculature results in excessive plateletaggregation, plaque and thrombus formation, atherosclerosis and stroke.In the context of the invention, the term “subject in need thereof” canbe an animal, mammal or human that is at risk for, or is alreadyexperiencing symptoms of the foregoing conditions. The collagen-inducedplatelet aggregation blocking ability of the F11R-antagonist peptides ofthe present invention provides heretofore unrecognized treatment andprevention options in subjects in need thereof, i.e., diseases anddisorders associated with excessive platelet aggregation.

The F11R-antagonist peptides of the present invention substantiallycorrespond to the amino acids of the N-terminus or first Ig domain ofhuman F11R.

A preferred F11R-antagonist peptide of the present invention is asequence of the N-terminal peptide of the F11R structure:SVTVHSSEPEVRIPENNPVKLSC (SEQ ID NO: 1).

Another preferred F11R-antagonist peptide of the present invention is asequence within the first Ig fold of the F11R structure: KSVTREDTGTYTC(SEQ ID NO: 4).

Homologs, analogs and fragments of these peptides which maintainF11-R-antagonist activity in a mammal, particularly humans are alsocontemplated by the present invention.

Another aspect of the present invention provides methods of interferingwith, blocking or otherwise preventing the interaction or binding ofplatelets to endothelial cells via F11R by employing theF11R-antagonists contemplated by the present invention.

The present invention also provides compositions for the treatment ofF11R-mediated disorders such as thrombosis, inflammatory thrombosis,atherosclerosis, atherosclerotic plaque formation, angiogenesis, heartattacks, hypertension, stroke and all other clinical disorders involvingthrombus formation, in animals, including humans and includes methods oftreating such disorders. The present invention is also directed to thetreatment and prevention of excessive bleeding following a wound injuryand inflammatory diseases of the nervous system. The compositionsinclude at least one of the F11R-antagonists, preferably at least oneF11R peptide antagonist according to the present invention, admixed witha pharmaceutically acceptable carrier.

In accordance with the present invention, the protein F11R serves asignificant role in the adhesion of platelets to inflamed endothelialcells. The present invention has identified that the activity of F11R iscritical for initiating platelet adherence to the vasculature with theformation of atherosclerotic plaques in blood vessels and for theformation of thrombi. Thus, any agents, chemicals or drugs that inhibitthe action of F11R (named here collectively: F11R-antagonists) willserve as powerful inhibitors of thrombus development in the circulation.Accordingly, the present invention provides F11R-antagonists as drugsuseful for the prevention and treatment of thrombosis, atherosclerosis,plaque formation, heart attacks, strokes, hypertension,immunothrombocytopenia, posttransfusion purpura, acute and chronicimmunothrombocytopenia, acquired disorders of platelet function,myeloproliferative disorders, uremia, liver disease, cardiopulmonarybypass, various types of thrombosis, inflammatory thrombosis, peripheralvein thrombosis, coronary artery thrombosis and other arterialthrombosis, atherosclerosis, disorders of angiogenesis, cancer growthand metastasis, and all other human disorders that involve angiogenesisand/or thrombus formation.

Platelet “aggregation” refers to any aggregative event between two ormore platelets. Thus, the term “platelet aggregation” encompassesplatelets aggregating with other platelets. The adherence or binding ofplatelets to something other than platelets is generally referred to bythe term “adhesion” or “binding.” Platelet adhesion may therefore referto platelet binding to collagen, basement membranes, cells expressingfunctional F11R on its cell surface either normally or due topathological inflammatory disease states, or by gene transfertechnology. For example, inflamed endothelial cells, which express anunmodified/wild type F11R or variants of the F11R polypeptide, mayparticipate in platelet adhesion. Adhesion of platelets may also beobserved by determining the F11R-mediated adhesion or binding of aplatelet to an artificial substrate or matrix comprising F11R orvariants thereof, as well as a platelet binding factor, protein,polypeptide, such as e.g., the extracellular domain of F11R, or animmobilized antibody directed to a platelet cell surface protein. Thesubstrate may be virtually any substrate used in binding assays, apreparation of cell membranes comprising F11R, or a monolayer of fixedF11R-expressing cells.

The term “proinflammatory” or “inflammation-inducing” refers to thetendency of an experimental or physiological stimulus to induce the EClayer of the endothelium to undergo biochemical or morphological changessimilar to those observed in actual pathological states or medicalconditions. One non-limiting example of such a stimulus is the effectresulting from contacting ECs with particular cytokines under suitableconditions. In such cases, the ECs acquire one or more characteristicsof an inflammatory state or an inflammatory condition. One relevanteffect of contacting ECs with one or more proinflammatory cytokines isthat the cellular localization of F11R on the EC surface is altered.Specifically, the pattern of F11R changes from its normal ornon-inflammatory state, characterized by F11R being concentrated at thejunctional regions, i.e., regions of cell-cell contact between ECs, to amore uniform distribution along the EC cell surface.

Terms used to describe diminished platelet aggregation and adhesion suchas “decreased,” “inhibited,” “impaired,” and the like, refer to loweredlevels of platelet aggregation or platelet adhesion without requiringany specific level of diminution, such as a complete or 100% inhibition.

One aspect of the invention is directed to methods of identifying acompound which prevents the adhesion of platelets to endothelial cellsand that inhibits platelet aggregation, also referred to as“F11R-antagonists.”

The term “compound” is taken to include both organic compounds such aspeptides, as well as inorganic compounds such as ion chelators oropiates. Antibodies, e.g., polyclonal or monoclonal antibodies directedagainst F11R, the Fab, Fab′, F(ab′)₂ fragments of such antibodies, aswell as single-chain anti-F11R antibodies can also be considered ascompounds useful in the present methods.

When applicable, an anti-F11R antibody (e.g., M.Ab.F11) orantigen-binding fragment thereof is a humanized antibody. A “humanizedantibody” is an antibody in which protein engineering is used to reducethe amount of ‘foreign’ protein sequence by substituting host antibody(e.g., mouse, rat) constant regions and the variable-domain frameworkregions with sequences that are found in human antibodies. It is furthercontemplated that when complete antibody molecules are to be employed,they may also be humanized or chimeric antibodies. Generally, a“chimeric antibody” is one where the constant regions of host origin,e.g., mouse or rat, are replaced by those of a human antibody.

Other preferred compounds include chemical compounds that can be derivedfrom the knowledge of the sequence of the F11R, from each of the abovesequences (i.e. SEQ ID NOS: 1-7) and from the combination of thesequences together. These include linear sequences, cyclic sequences,annealing of the peptides together (preferably SEQ ID NOS: 1 and 4), andany other possible derivations using standard peptide chemistrytechniques. In one embodiment the present invention contemplates anycompound whose structure is based on the interaction of peptides 1 and 4(SEQ ID NOS. 1 and 4), which form the binding site of the mature humanplatelet F11R.

As used herein a “mimetic” or “peptidomimetic” of a compound'sfunctional site refers to a compound in which chemical structures ofprotein or peptide sequences necessary for functional activity of acompound's functional site have been replaced with other chemicalstructures that mimic the conformation of the functional site. Anexample of a peptidomimetic contemplated by the present inventionincludes a compound (e.g. a small organic molecule) including portionswith residues which interact sterically with the binding site of theF11R molecule. In accordance with the present invention, F11Rpeptidomimetic drugs can be designed on the basis of, for example,peptides having SEQ ID NOs. 1 and 4 and the tertiary structure of thebinding site of F11R, including parts of the protein containing thesesequences. Such peptidomimetic drugs with structural relationshipsanalogous to that observed between morphine, enkephalins andbeta-endorphins, are suitable as therapeutic agents. The design andsynthesis of peptidomimetic molecules continues to be at the forefrontof drug design and discovery and many peptidomimetic frameworks andmethods for their synthesis have been developed (Babine, R. E.; Bender,S. L., Chem. Rev., 97:1359, 1997; Hanessian, S.; et al., Tetrahedron,53:12789, 1997; Fletcher, M. D.; Cambell, M. C., Chem. Rev., 98:763,1998), these teachings are incorporated herein by reference.

The peptidomimetics in accordance with the present invention can bedeveloped, for example, with the aid of computerized molecular modeling.In a preferred embodiment, the present invention provides apharmaceutical composition comprising SEQ ID NO: 1 or SEQ ID NO: 4wherein SEQ ID NO: 1 or SEQ ID NO: 4 comprises peptidomimetics that arecapable of specific binding with the F11R binding site. Peptide mimeticsthat are structurally similar to therapeutically useful peptides can beused to produce an equivalent therapeutic or prophylactic effect.Generally, peptidomimetics are structurally similar to a paradigmpolypeptide (i.e., a polypeptide that has a biochemical property orpharmacological activity), such as SEQ ID NO: 1 or SEQ ID NO: 4, buthave one or more peptide linkages optionally replaced by a linkageselected from the group consisting of: —CH₂NH—, —CH₂S—, —CH₂CH₂—,—CH═CH—(cis and trans), —COCH₂—, —CH(OH)CH₂—, and —CH₂SO—, by methodsknown in the art and further described in the following references:Spatola, A. F. in “Chemistry and Biochemistry of Amino Acids, Peptides,and Proteins,” B. Weinstein, eds., Marcel Dekker, New York, p. 267(1983); Spatola, A. F., Vega Data (March 1983), Vol. 1, Issue 3,“Peptide Backbone Modifications” (general review); Morley, J. S., TrendsPharm Sci (1980) pp. 463-468 (general review); Hudson, D. et al., (1979)Int J Pept Prot Re 14:177-185 (—CH₂NH—, —CH₂ —CH₂—); Spatola, A. F. etal., (1986) Life Sci 38:1243-1249 (—CH₂S); Hann, M. M., (1982) J ChemSoc Perkin Trans 1307-314 (—CH═CH—, cis and trans); Almquist, R. G. etal., (1980) J Med Chem 23: 1392-1398 (—COCH₂—); Jennings-White, C. etal., (1982) Tetrahedron Lett 23:2533 (—COCH₂—); Szelke, M. et al.,European Appln. EP 45665 (1982) CA: 97:39405 (1982) (—CH(OH)CH₂—);Holladay, M. W. et al., (1983) Tetrahedron Lett 24:4401-4404(—C(OH)CH₂—); and Hruby, V. J., (1982) Life Sci 31:189-199 (—CH₂S—);each of which is incorporated herein by reference.

In another embodiment, a particularly preferred non-peptide linkage is—CH₂NH—. Such peptidomimetics may have significant advantages overpolypeptide embodiments, including, for example: more economicalproduction, greater chemical stability, enhanced pharmacologicalproperties (half-life, absorption, potency, efficacy, etc.), alteredspecificity (e.g., a broad-spectrum of biological activities), reducedantigenicity, and others.

A variety of designs for peptidomimetics are possible. For example,cyclic peptides, in which the necessary conformation for binding isstabilized by nonpeptides, are specifically contemplated. U.S. Pat. No.5,192,746 to Lobl, et al., U.S. Pat. No. 5,169,862 to Burke, Jr., et al,U.S. Pat. No. 5,539,085 to Bischoff, et al., U.S. Pat. No. 5,576,423 toAversa, et al., U.S. Pat. No. 5,051,448 to Shashoua, and U.S. Pat. No.5,559,103 to Gaeta, et al., all hereby incorporated by reference,describe multiple methods for creating such compounds. Synthesis ofnonpeptide compounds that mimic peptide sequences is also known in theart. Eldred, et al., (J. Med. Chem. 37:3882 (1994)) describe nonpeptideantagonists that mimic the peptide sequence. Likewise, Ku, et al., (J.Med. Chem. 38:9 (1995)) give further elucidation of the synthesis of aseries of such compounds. Derivatives of e.g. SEQ. ID. NO.: 1 or SEQ IDNO: 4 can be produced using recombinant nucleic acid moleculetechniques.

Modifications to a specific peptide may be deliberate, as throughsite-directed mutagenesis and amino acid substitution duringbiosynthesis, or may be accidental such as through mutations in hosts,which produce the peptide. Peptides including derivatives can beobtained using standard mutagenesis techniques such as those describedin Sambrook et al., Molecular Cloning, Cold Spring Harbor LaboratoryPress (1989). For example, Chapter 15 of Sambrook describes proceduresfor site-directed mutagenesis of cloned DNA. Derivatives of SEQ ID NOs.:1 and 4 include, but are not limited by modification occurring during orafter translation, for example, by phosphorylation, glycosylation,crosslinking, acylation, proteolytic cleavage, linkage to a therapeuticprotein, an antibody molecule, membrane molecule or other ligand (seeFerguson et al., 1988, Annu. Rev. Biochem. 57:285-320). Specific typesof genetically produced derivatives also include, but not limited byamino acid alterations such as deletions, substitutions, additions, andamino acid modifications. A “deletion” refers to the absence of one ormore amino acid residue(s) in the related peptide. An “addition” refersto the presence of one or more amino acid residue(s) in the relatedpeptide. Additions and deletions to a peptide may be at the aminoterminus, the carboxy terminus, and/or internal, can be produced bymutation in e.g., SEQ ID NO: 1 encoding DNA and/or by peptidepost-translation modification. Amino acid “modification” refers to thealteration of a naturally occurring amino acid to produce anon-naturally occurring amino acid. Analogs of e.g. SEQ ID NO: 1 withunnatural amino acids can be created by site-specific incorporation ofunnatural amino acids into polypeptides during the biosynthesis, asdescribed in Christopher J. Noren, Spencer J. Anthony-Cahill, Michael C.Griffith, Peter G. Schultz, 1989 Science, 244: 182-188. A “substitution”refers to the replacement of one or more amino acid residue(s) byanother amino acid residue(s) in the peptide. Mutations can be made ine.g., SEQ ID NO: 1 encoding DNA such that a particular codon is changedto a codon, which codes for a different amino acid. Such a mutation isgenerally made by making the fewest nucleotide changes possible. Asubstitution mutation of this sort can be made to change an amino acidin the resulting peptide in a non-conservative manner (i.e., by changingthe codon from an amino acid belonging to a grouping of amino acidshaving a particular size or characteristic to an amino acid belonging toanother grouping) or in a conservative manner (i.e., by changing thecodon from an amino acid belonging to a grouping of amino acids having aparticular size or characteristic to an amino acid belonging to the samegrouping). Such a conservative change generally leads to less change inthe structure and function of the resulting peptide. To some extent thefollowing groups contain amino acids which are interchangeable: thebasic amino acids lysine, arginine, and histidine; the acidic aminoacids aspartic and glutamic acids; the neutral polar amino acids serine,threonine, cysteine, glutamine, asparagine and, to a lesser extent,methionine; the nonpolar aliphatic amino acids glycine, alanine, valine,isoleucine, and leucine (however, because of size, glycine and alanineare more closely related and valine, isoleucine and leucine are moreclosely related); and the aromatic amino acids phenylalanine,tryptophan, and tyrosine. In addition, although classified in differentcategories, alanine, glycine, and serine seem to be interchangeable tosome extent, and cysteine additionally fits into this group, or may beclassified with the polar neutral amino acids. Although proline is anonpolar neutral amino acid, its replacement represents difficultiesbecause of its effects on conformation. Thus, substitutions by or forproline are not preferred, except when the same or similarconformational results can be obtained. The conformation conferringproperties of proline residues may be obtained if one or more of theseis substituted by hydroxyproline (Hyp). Derivatives can containdifferent combinations of alterations including more than one alterationand different types of alterations.

The ability of the derivative to retain some activity can be measuredusing techniques described herein and/or using techniques known to thoseskilled in the art for measuring the F11R receptor binding activity.“Derivatives” of e.g., SEQ. ID. NO.: 1 are functional equivalents havingsimilar amino acid sequence and retaining, to some extent, theactivities of SEQ ID NO: 1. By “functional equivalent” is meant thederivative has an activity that can be substituted for the activity ofSEQ. ID NO:1. Preferred functional equivalents retain the full level ofF11R-binding activity as measured by assays known to these skilled inthe art. Preferred functional equivalents have activities that arewithin 1% to 10,000% of the activity of e.g., SEQ ID NO: 1, morepreferably between 10% to 1000%, and more preferably within 50% to 200%.Derivatives have at least 50% sequence similarity, preferably 70%, morepreferably 90%, and even more preferably 95% sequence similarity to SEQ.ID. NO: 1. “Sequence similarity” refers to “homology” observed betweenamino acid sequences in two different polypeptides, irrespective ofpolypeptide origin. A “residue” refers to an amino acid incorporated inthe peptide by an amide bond, for example. Approaches to designingpeptide mimetics are known in the art. For example, see Farmer, P. S. inDrug Design (E. J. Ariens, ed). Academic Press, New York, 1980, vol. 10,pp. 119-143; Ball J. B. and Alewood, P.F. (1990) J. Mol. Recognition.3:55, Morgan B.A. and Ganor, J.A. (1985) Ann. Rev. Med. Chem. 24:243 andFreidinger R. M. (1989) Trends Pharmacol. Sci: 10:270, incorporatedherein by reference. In one embodiment, the present inventioncontemplates all peptidomimetics which can be designed based on theknowledge of the sequence and of the three-dimensional structure of theF11R molecule of the invention including but not limited to all mimeticcompounds which can be conventionally synthesized by an ordinarilyskilled chemist to bind to, antagonize, act as an agonist, inhibit,promote, block, or otherwise functionally interact with the binding siteof the F11R.

Most preferred compounds of the present methods are peptides which aremade to resemble the monoclonal antibody F11 (“M.Ab.F11”) binding siteon platelets.

In one example a peptidomimetic entitled 4D may be used. Peptidomimetic4D is based on the amino acid sequence of the peptide of SEQ ID NO: 4.SEQ ID NO: 4 is a 13 amino acid sequence located within the firstimmunoglobulin fold of the F11R protein. A 13-mer peptide with thissequence is an inhibitor of the biological activities of F11R measuredboth in vitro and in cell cultures. A non-natural peptide that mimicsthe structure of the peptide of SEQ ID NO: 4 can be synthesized withsubstituted D-amino acids in the two main sites for cleavage byendopeptidases trypsin and with the carboxyl group amidated (CONH₂) forthe prevention of dimeric-disulphides. This modification resulted in thedesign of a mimetic-peptide referred to herein as peptide 4D. Thesequence of F11R peptide 4D is dKSVTdREDTGTYTC-CONH₂ (SEQ ID NO: 10),with the underlined letters indicating the positions of the twosubstituted D-amino acids. Use of peptide 4D is further described inExample 9 below.

Other peptidomimetics can also be used. In another example based on SEQID NO: 4, the sequence of other F11R peptides can be, among others,KSVTdREDTGTYTC-CONH₂ (SEQ ID NO: 11), SVTdREDTGTYTC-CONH₂ (SEQ ID NO:12), VTdREDTGTYTC-CONH₂ (SEQ ID NO: 13), TdREDTGTYTC-CONH₂ (SEQ ID NO:14), dREDTGTYTC-CONH₂ (SEQ ID NO: 15), REDTGTYTC-CONH₂ (SEQ ID NO: 16),EDTGTYTC-CONH₂ (SEQ ID NO: 17), DTGTYTC-CONH₂ (SEQ ID NO: 18),TGTYTC-CONH₂ (SEQ ID NO: 19) or GTYTC-CONH₂ (SEQ ID NO: 20). In otherexamples, the sequence of other F11R peptides can be, among others,dKSVTdREDTGTYTC (SEQ ID NO: 21), dKSVTdREDTGTYT (SEQ ID NO: 22),dKSVTdREDTGTY (SEQ ID NO: 23), dKSVTdREDTGT (SEQ ID NO: 24), dKSVTdREDTG(SEQ ID NO: 25), dKSVTdREDT (SEQ ID NO: 26), dKSVTdRED (SEQ ID NO: 27),dKSVTdRE (SEQ ID NO: 28), dKSVTdR (SEQ ID NO: 29), dKSVTd (SEQ ID NO:30) or dKSVT (SEQ ID NO: 31).

In another example, a peptidomimetic entitled 1P may be used.Peptidomimetic 1P is based on the amino acid sequence of the peptide ofSEQ ID NO: 1. SEQ ID NO: 1 is a 23 amino acid sequence located withinthe N-terminus of the F11R protein. Peptidomimetic 1P can be manyvariations including, but not limited to, VTVHSSEPEVRIPENNPVKLSC (SEQ IDNO: 32), TVHSSEPEVRIPENNPVKLSC (SEQ ID NO: 33), VHSSEPEVRIPENNPVKLSC(SEQ ID NO: 34), HSSEPEVRIPENNPVKLSC (SEQ ID NO: 35), SSEPEVRIPENNPVKLSC(SEQ ID NO: 36), SEPEVRIPENNPVKLSC (SEQ ID NO: 37), EPEVRIPENNPVKLSC(SEQ ID NO: 38), PEVRIPENNPVKLSC (SEQ ID NO: 39), EVRIPENNPVKLSC (SEQ IDNO: 40), VRIPENNPVKLSC (SEQ ID NO: 41), RIPENNPVKLSC (SEQ ID NO: 42),IPENNPVKLSC (SEQ ID NO: 43), PENNPVKLSC (SEQ ID NO: 44), ENNPVKLSC (SEQID NO: 45), NNPVKLSC (SEQ ID NO: 46), NPVKLSC (SEQ ID NO: 47), PVKLSC(SEQ ID NO: 48) or VKLSC (SEQ ID NO: 49). Other variations ofpeptidomimetic 1P include, but are not limited to,SVTVHSSEPEVRIPENNPVKLS (SEQ ID NO: 50), SVTVHSSEPEVRIPENNPVKL (SEQ IDNO: 51), SVTVHSSEPEVRIPENNPVK (SEQ ID NO: 52), SVTVHSSEPEVRIPENNPV (SEQID NO: 53), SVTVHSSEPEVRIPENNP (SEQ ID NO: 54), SVTVHSSEPEVRIPENN (SEQID NO: 55), SVTVHSSEPEVRIPEN (SEQ ID NO: 56), SVTVHSSEPEVRIPE (SEQ IDNO: 57), SVTVHSSEPEVRIP (SEQ ID NO: 58), SVTVHSSEPEVRI (SEQ ID NO: 59),SVTVHSSEPEVR (SEQ ID NO: 60), SVTVHSSEPEV (SEQ ID NO: 61), SVTVHSSEPE(SEQ ID NO: 62), SVTVHSSEP (SEQ ID NO: 63), SVTVHSSE (SEQ ID NO: 64),SVTVHSSE (SEQ ID NO: 65), SVTVHSS (SEQ ID NO: 66), SVTVHS (SEQ ID NO:66) and SVTVH (SEQ ID NO: 67).

“F11R” refers to a receptor protein on the surface of human platelets asa target for a stimulatory M.Ab.F11. “F11R” is also referred to as humanortholog of the murine protein called junctional adhesion molecule(JAM), specifically named JAM-1 and JAM-A. F11R from either platelets orendothelial cells comprises an extracellular domain consisting of twoIg-folds, a transmembrane domain and a short cytoplasmic portion. ThecDNA encoding the F11R can be engineered, e.g., to delete thetransmembrane and cytoplasmic domain thereby providing a polynucleotideencoding the extracellular domain. Expression of the F11R extracellulardomain in eukaryotic cells results in its synthesis and secretion,thereby indicating that it is a soluble polypeptide.

“F11R antagonists” and “F11R antagonist peptides” further refers to anycompound that can bind to the active site of the F11R protein,specifically, but not limited to a pocket formed by the N-terminal 23amino acid region and 13 amino acid region in the first Ig fold. By suchbinding, the action of F11R is inhibited, i.e. alignment of plateletsand endothelial cells in F11R-mediated trans-homophilic interactionthrough the steric pocket, as depicted in FIG. 1, is blocked so thatplatelet aggregation or thrombosis, atherosclerosis, heart attacks,strokes, and all other human disorders that involve thrombus formation,can be prevented or treated. By “F11R antagonist peptide” is also meanta peptide that inhibits, suppresses or causes the cessation of at leastone F11R mediated biological activity by e.g. interfering with orotherwise preventing the interaction or binding of platelets toendothelial cells and thereby inhibit platelet aggregation orinterfering with the role of some protein in angiogenesis and thuspreventing the growth of tumors.

In accordance with the present invention, two peptide sequences of theF11R have been determined the sequences correspond to (the N-terminusSVTVHSSEPEVRIPENNPVKLSC (SEQ ID NO: 1), and the first Ig fold sequenceKSVTREDTGTYTC (SEQ ID NO: 4). The peptide sequences of the presentinvention inhibit the adhesion of platelets to endothelial cells andinhibit platelet aggregation.

As used herein, “peptide” refers to a linear series of amino acidresidues linked to one another by peptide bonds between the alpha-aminoand carboxy groups of adjacent amino acid residues. The term “syntheticpeptide” is intended to refer to a chemically derived chain of aminoacid residues linked together by peptide bonds. The term “syntheticpeptide” is also intended to refer to recombinantly produced peptides inaccordance with the present invention. According to the presentinvention, preferred F11R antagonists include peptides (referred toherein as “F11R antagonist peptides”) and antibodies. Additionally,analogs, homologs and fragments of the novel peptides provided hereinare included within the scope of the term “F11R antagonist peptide”.

By “homologs” is meant the corresponding peptides from F11R proteins ofother mammalian species substantially homologous at the overall protein(i.e., mature protein) level to human F11R, so long as such homologouspeptides retain the F11R antagonist activity.

By “analogs” or “F11R-Antagonist Peptide Analysis” is meant peptideswhich differ by one or more amino acid alterations, which alterations,e.g., substitutions, additions or deletions of amino acid residues, donot abolish the F11R antagonist properties of the relevant peptides.Thus, an analog can comprise a peptide having a substantially identicalamino acid sequence to a peptide provided herein and in which one ormore amino acid residues have been conservatively or non-conservativelysubstituted. Examples of conservative substitutions include thesubstitution of a non-polar (hydrophobic) residue such as isoleucine,valine, leucine or methionine for another Likewise, the presentinvention contemplates the substitution of one polar (hydrophilic)residue such as between arginine and lysine, between glutamine andasparagine, and between glycine and serine. Additionally, thesubstitution of a basic residue such as lysine, arginine or histidinefor another or the substitution of one acidic residue such as asparticacid or glutamic acid for another is also contemplated. Examples ofnon-conservative substitutions include the substitution of a non-polar(hydrophobic) residue such as isoleucine, valine, leucine, alanine,methionine for a polar (hydrophilic) residues such as cyteine,glutamine, glutamic acid, lysine and/or a polar residue for a non-polarresidue.

The phrase “conservative substitution” also includes the use ofchemically derivatized residues in place of a non-derivatized residuesas long as the peptide retains the requisite F11R antagonist, inhibitionproperties as conventionally measured. Analogs also include the presenceof additional amino acids or the deletion of one or more amino acidswhich do not affect F11R-mediated biological activity. For example,analogs of the subject peptides can contain an N- or C-terminalcysteine, by which, if desired, the peptide can be covalently attachedto a carrier protein, e.g., albumin. Such attachment, it is believed,will minimize clearing of the peptide from the blood and also preventproteolysis of the peptides. In addition, for purposes of the presentinvention, peptides containing D-amino acids in place of L-amino acidsare also included in the term “conservative substitution”. The presenceof such D-isomers can help minimize proteolytic activity and clearing ofthe peptide.

The term “fragment” refers to any subject peptide having an amino acidsequence shorter than that of any peptide depicted in SEQ ID NOS: 1-5and 7 and which fragment retains the F11R-mediated antagonist activityof the subject peptides.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of synthetic organic chemistry,protein chemistry, molecular biology, microbiology, and recombinant DNAtechnology, which are well within the skill of the art. These techniquesare applied in connection with peptide synthesis, recombinant productionof peptides and peptide mutagenesis, for example. Such techniques areexplained fully in the literature. See e.g., Scopes, R. K., ProteinPurification Principles and Practices, 2d ed. (Springer-Verlag. 1987),Methods in Enzymology (M. Deutscher, ed., Academic Press, Inc. 1990),Sambrook, et al., Molecular Cloning: A laboratory Manual, 2d ed., (ColdSpring Harbor Press, Cold Spring Harbor, N.Y., 1989), Handbook ofExperimental Immunology, Vols. I-IV (D. M. Weir and C. C. Blackwell,eds., 1986, Blackwell Scientific Publications), House, Modern SyntheticReactions, 2d ed., (Benjamin/Cummings, Menlo Park, Cal., 1972).

The peptides of the present invention, homologs, analogs and fragmentsthereof can be synthesized by a number of known techniques. For example,the peptides may be prepared using the solid-phase synthetic techniqueinitially described by Merrifield, in J. Am. Chem. Soc. 85:2149-2154(1963). Other peptide synthesis techniques can be found in M. Bodanszky,et al. Peptide Synthesis, John Wiley & Sons, 2d Ed., (1976) and otherreferences readily available to those skilled in the art. A summary ofpolypeptide synthesis techniques can be found in J. Stuart and J. D.Young, Solid Phase Peptide Synthesis, Pierce Chemical Company, Rockford,Ill., (1984). Peptides may also be synthesized by solution methods asdescribed in The Proteins, Vol. II. 3d Ed., Neurath, H. et al., Eds., p.105-237, Academic Press, New York, N.Y. (1976). Appropriate protectivegroups for use in different peptide syntheses are described in theabove-mentioned texts as well as in J. F. W. McOmie, Protective Groupsin Organic Chemistry, Plenum Press, New York, N.Y. (1973). The peptidesof the present invention can also be prepared by chemical or enzymaticcleavage from larger portions of the F11R molecule or from the entireF11R molecule.

Additionally, the peptides of the present invention can also be preparedby recombinant DNA techniques (see e.g. Current Protocols in MolecularCloning Ausubel et al., 1995, John Wiley & Sons, New York); Sambrook etal., 1989, Molecular Cloning: A Laboratory Manual, Second Edition, ColdSpring Harbor Laboratory Press, New York; Coligan et al. CurrentProtocols in Immunology, John Wiley & Sons Inc., New York, N.Y. (1994)).The skilled artisan understands that any of a wide variety of expressionsystems can be used to provide the recombinant peptides of the presentinvention. The precise host cell used is not critical to the invention.The F11R antagonist peptides can be produced in a prokaryotic host (e.g.E. coli), or in a eukaryotic host (e.g., S. cerevisiae or mammaliancells, e.g. COS 1, CHO, NIH3T3, and JEG3 cells, or in the cells of anarthropod, e.g. S. frugiperda). Such cells are available from e.g. theAmerican Type Culture Collection, Manassas, Va. The method oftransfection and the choice of expression vehicle will depend on thehost system selected. Transformation and transfection methods aredescribed, e.g. in Sambrook et al. supra; expression vehicles can bechosen from those provided e.g. in Cloning Vectors: A Laboratory ManualP. H. Powels et al (1985), Supp. 1987.

For most of the amino acids used to build proteins, more than one codingnucleotide triplet (codon) can code for a particular amino acid residue.This property of the genetic code is known as redundancy. Therefore, anumber of different nucleotide sequences can code for a particularsubject F11R antagonist peptide. The present invention also contemplatesa deoxyribonucleic acid (DNA) molecule or segment that defines a genecoding for, i.e., capable of expressing, a subject peptide or a subjectchimeric peptide from which a peptide of the present invention can beenzymatically or chemically cleaved. DNA molecules that encode peptidesof the present invention can be synthesized by chemical techniques, forexample, the phosphotriester method of Matteuccie, et al., J. Am. Chem.Soc. 103:3185 (1981). Using a chemical DNA synthesis technique, desiredmodifications in the peptide sequence can be made by makingsubstitutions for bases which code for the native amino acid sequence.Ribonucleic acid equivalents of the above described DNA molecules mayalso be used.

A nucleic acid molecule comprising a vector capable of replication andexpression of a DNA molecule defining coding sequence for a subjectpolypeptide or subject chimeric polypeptide is also contemplated.

The peptides of the present invention are chemically synthesized byconventional techniques such as the Merrifield solid phase technique. Ingeneral, the method comprises the sequential addition of one or moreamino acid residues to a growing peptide chain. Normally, either theamino or carboxyl group of the first amino acid residue is protected bya suitable, selectively removable protecting group. A different,selectively removable protecting group is utilized for amino acidscontaining a reactive side group such as lysine.

A preferred method of solid phase synthesis entails attaching theprotected or derivatized amino acid to an inert solid support throughits unprotected carboxyl or amino group. The protecting group of theamino or carboxyl group is then selectively removed and the next aminoacid in the sequence having the complementary (amino or carboxyl) groupsuitably protected is admixed and reacted under conditions suitable forforming the amide linkage with the residue already attached to the solidsupport. The protecting group of the amino carboxyl group is thenremoved from this newly added amino acid residue, and the next aminoacid (suitably protected) is then added, and so forth. After all thedesired amino acids have been linked in the proper sequence, anyremaining terminal and side group protecting groups including the solidsupport are removed sequentially or concurrently to yield the finalpeptide. The lyophilized oligopeptides are resuspended in doubledistilled H₂O at 2 mg/ml as stock solutions and subsequently diluted inM199-HPS for experiments. Consistent with the observed properties of thepeptides of the invention, the present peptides can be used to inhibit,suppress, or cause the cessation of at least one F11R-mediatedbiological activity. F11R functions in the biochemical events associatedwith platelets aggregation and adhesion of platelets to endothelialcells. Accordingly, the present invention contemplates methods to block,interrupt or otherwise prevent the association of platelets toendothelial cells and thereby effectively treat or prevent F11R-cellassociated disorders such as thrombosis, for example.

F11R-mediated disorders such as, for example, thrombosis,atherosclerosis, heart attacks and strokes are F11R-dependent thereforetreatable with the F11R antagonists, preferably F11R antagonist peptidesor peptidomimetics of the present invention. Other F11R related diseasesare also contemplated by the present invention.

In another embodiment of the present invention, one or more F11Rantagonists, e.g., F11R antagonist peptides, peptidomimetics orantibodies, are included in pharmaceutical compositions.

Preferably, compositions containing the F11R antagonist peptides orpeptidomimetics of the present invention are administered intravenouslyto inhibit, suppress, or cause the cessation of at least oneF11R-mediated biological activity. When administered intravenously, thepeptide compositions can be combined with other ingredients, such ascarriers and/or adjuvants. The peptides may also be covalently attachedto a protein carrier, such as albumin, so as to minimize clearing of thepeptides. There are no limitations on the nature of the otheringredients, except that such ingredients must be pharmaceuticallyacceptable, efficacious for their intended administration and cannotdegrade the activity of the active ingredients of the compositions.Examples of other anti-inflammatory ingredients contemplated by thepresent invention include, but are not limited to anti-F11R antibodies,NSAIDS, steroids, or cyclosporin-A. When employed together with F11Rantagonists, these agents may be employed in lesser dosages than whenused alone.

The pharmaceutical forms suitable for injection include sterile aqueoussolutions or dispersions and sterile powders for the extemporaneouspreparation of sterile injectable solutions or dispersions. In all casesthe ultimate solution form must be sterile and fluid. Typical carriersinclude a solvent or dispersion medium containing, for example, waterbuffered aqueous solutions (i.e., biocompatible buffers), ethanol,polyols such as glycerol, propylene glycol, polyethylene glycol,suitable mixtures thereof, surfactants or vegetable oils. Sterilizationcan be accomplished by any art-recognized technique, including but notlimited to, filtration or addition of antibacterial or antifungalagents, for example, paraben, chlorobutano, phenol, sorbic acid orthimerosal. Further, isotonic agents such as sugars or sodium chloridecan be incorporated in the subject compositions.

Production of sterile injectable solutions containing the subjectpeptides is accomplished by incorporated these compounds in the requiredamount in the appropriate solvent with various ingredients enumeratedabove, as required, followed by sterilization, preferably filtersterilization. To obtain a sterile powder, the above solutions arevacuum-dried or freeze-dried as necessary. Further to administration byinjection intravenously, peptides may also be administered parenterallyintramuscularly, intraperitoneally, intrathecally, in a suppository,transdermally, topically, or orally.

When the peptides or peptidomimetics of the invention are administeredorally, the pharmaceutical compositions thereof containing an effectivedose of the peptide can also contain an inert diluent, as assimilableedible carrier and the like, be in hard or soft shell gelatin capsules,be compressed into tablets, or can be in an elixir, suspension, syrup orthe like.

The subject peptides or peptidomimetics are thus compounded forconvenient and effective administration in pharmaceutically effectiveamounts with a suitable pharmaceutically acceptable carrier in atherapeutically effective dose.

The peptides and peptidomimetics should preferably be administered in anamount of at least about 50 mg per dose, more preferably in an amount upto about 500 mg to about 1 gram per dose. Since the peptide compositionsof this invention will eventually be cleared from the bloodstream,re-administration of the compositions is indicated and preferred.

The peptides and peptidomimetics can be administered in a mannercompatible with the dosage formulation and in such amount as well betherapeutically effective. Systemic dosages depend on the age, weightand conditions of the patient and on the administration route. Forexample, a suitable dose for the administration to adult humans rangesfrom about 1 mg/kg of body weight about 10 mg per kilogram of bodyweight. The present invention also contemplates that the peptide orpeptidomimetic compositions can be suitably coated on stents, lines, andtubes with a therapeutically effective amount of the peptide whichamount can be readily determined by the skilled practitioner.

As used herein, a pharmaceutically acceptable carrier includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic agents the like. The use of such media and agents arewell-known in the art. The pharmaceutically acceptable carriers used inconjunction with the peptides of the present invention vary according tothe mode of administration. For example, the compositions can beformulated in any suitable carrier for oral liquid formulation such assuspensions, elixirs and solutions. Compositions for liquid oral dosageinclude any of the usual pharmaceutical media such as, for example,water, oils, alcohols, flavoring agents, preservatives, coloring agentsand the like. In the case of oral solid preparations (capsules andtablets) carriers such as starches, sugars, diluents, granulatingagents, lubricants, binders, disintegrating agents and the like can beused. In addition, carriers such as liposomes and microemulsions can beused.

In a further aspect of the present invention, the pharmaceuticalcompositions of the present invention are employed for the treatment ofF11R mediated pathological disorders. Thus, the present inventionprovides methods of treating an F11R mediated disorder in a subject byadministering a therapeutically effective amount of a pharmaceuticalcomposition of the present invention.

The term “therapeutically effective amount” means the dose required totreat an F11R-mediated disorder.

By “an F11R-mediated disorder” is meant a pathological disorder, theonset, progression or the persistence of the symptoms of which requiresthe participation of F11R molecules. Particularly, F11R-mediateddisorders contemplated by the present invention include thrombosis,atherosclerosis, heart attacks and strokes. In addition, the inventorshave determined that collagen-induced platelet aggregation and secretioncan be blocked completely by the F11R peptides of the present invention.

Accordingly, “an F11R-mediated disorder” also contemplates excessivebleeding as may occur following a wound injury. Furthermore, and inaccordance with the present invention “an F11R-mediated disorder” caninclude inflammatory diseases of the nervous system.

The term “treatment” or “treat” refers to effective inhibition,suppression or cessation of the F11R activity so as to prevent or delaythe onset, retard the progression or ameliorate the symptoms of thedisorder.

The term “subject” refers to any mammalian subject. Preferably, thesubject is a human.

The present invention thus provides methods of interfering with,blocking or otherwise preventing the interaction or binding of plateletswith endothelial cells by employing the F11R antagonists contemplated bythe present invention.

The F11R antagonist peptides of the present invention (or homologs,analogs or fragments) can be used to raise single-chain antibodies (SAb)or humanized monoclonal antibodies useful in the invention. The peptidescan be coupled to a carrier protein such as KLH as described in Ausubelet al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons,New York. The KLH-antagonist peptide is mixed with Freund's adjuvant andinjected into guinea pigs, rats, donkeys and the like or preferably intorabbits. Antibodies can be purified by peptide antigen affinitychromatography.

A single-chain antibody (SAb) is created by fusing together the variabledomains of the heavy and light chains using a short peptide linker,thereby reconstituting an antigen binding site on a single molecule.Such single-chain antibody variable fragments (Fvs) can be fused to allor a portion of the constant domains of the heavy chain of animmunoglobulin molecule, if necessary. The use of sAb avoids thetechnical difficulties in the introduction of more than one geneconstruct into host cells. Single chain antibodies and methods for theirproduction are known in the art. See, e.g., Bedzyk et al. (1990) J.Biol. Chem., 265:18615; Chaudhary et al. (1990) Proc. Natl. Acad. Sci.,87:9491; U.S. Pat. No. 4,946,778 to Ladner et al.; and U.S. Pat. No.5,359,046 to Capon et al.

Monoclonal antibodies can be prepared using F11R antagonist peptides andstandard hybridoma technology (see e.g. Kohler et al., (1975) Nature256:495; Hammerling et al., (1981) In Monoclonal Antibodies and T CellHybridomas, Elsevier, New York). For example, monoclonal antibodies toF11R antagonist peptides (homologs, analogs or fragments thereof) can beraised in Balb/C or other similar strains of mice by immunization withpurified or partially purified preparations of F11R antagonist peptides.The spleens of the mice can be removed, and their lymphocytes fused to amouse myeloma cell line. After screening of hybrids by known techniques,a stable hybrid will be isolated that produces antibodies against F11Rantagonist peptides. The monoclonal antibody can be examined for itsability to inhibit the biological activity of F11R, e.g. plateletaggregation. Once produced, monoclonal antibodies are tested forspecific F11R recognition by Western blot or immunoprecipitationanalysis (by methods described in Ausubel et al., supra). Antibodieswhich antagonize F11R/platelet aggregation are considered to be usefulantagonists in the invention.

The monoclonal antibodies of the present invention can be humanized toreduce the immunogenicity for use in humans. One approach is to makemouse-human chimeric antibodies having the original variable region ofthe murine mAb, joined to constant regions of a human immunoglobulin.Chimeric antibodies and methods for their production are known in theart. See, e.g., Cabilly et al., European Patent Application 125023(published Nov. 14, 1984); Taniguchi et al., European patent Application171496 (published Feb. 19, 1985); Morrison et al., European PatentApplication 173494 (published Mar. 5, 1986); Neuberger et al., PCTApplication WO 86/01533, (published Mar. 13, 1986); Kudo et al.,European Patent Application 184187 (published Jun. 11, 1986); Robinsonet al., International Patent Publication #PCT/US86/02269 (published 7May 1987); Liu et al., Proc. Natl. Acad. Sci. USA 84:3439-3443 (1987);Sun et al., Proc. Natl. Acad. Sci. USA 84:214-218 (1987); Better et al.,Science 240:1041-1043 (1988). These references are incorporated hereinby reference. Generally, DNA segments encoding the H and L chainantigen-binding regions of the murine mAb can be cloned from themAb-producing hybridoma cells, which can then be joined to DNA segmentsencoding C_(H) and C_(L) regions of a human immunoglobulin,respectively, to produce murine-human chimeric immunoglobulin-encodinggenes.

The invention is further illustrated by the following specific exampleswhich are not intended in any way to limit the scope of the invention.

Example 1 Human Platelets

Whole blood was collected into the anticoagulant ACD (pH 4.6), asdetailed (Kornecki et al. (1990) J. Biol. Chem., 265:10042-10048,incorporated herein by reference). Platelets were washed and isolated bydifferential centrifugation, and resuspended in a Tyrode-albumin (0.35%)solution buffered with 11.9 mM sodium bicarbonate (pH 7.35) in thepresence of apyrase, heparin, and PGE₁ (Id.). Final platelet suspensionsdid not contain any inhibitors. Platelet aggregation was measured in aChronolog Whole Blood Lumi-Aggregometer (Chronolog Corp. Havertown,Pa.). Potentiation of platelet aggregation was measured by adding amixture of two platelet agonists, each at a sub-threshold concentrationthat did not induce any platelet aggregation. The lowest concentrationof each agonist which caused platelet aggregation was determined inthese experiments for each donor on the day of blood collection.

Immunoblotting.

Polyacrylamide gel electrophoresis, immunoblotting of transferredproteins onto nitrocellulose strips, and detection using ECLchemluminescence were performed as detailed (Kornecki et al. (1990) J.Biol. Chem., 265:10042-10048).

Antibodies.

Monoclonal antibody M.Ab.F11 (IgG1 isotype) was affinity-purified asdescribed (Kornecki et al. (1990) J. Biol. Chem., 265:10042-10048).Histidine antibody was obtained from InVitrogen (Carlsbad, Calif.).

Construction of the Plasmid pcDNA3.1/F11R.

A 726-base-pair fragment (nucleotide-6 till+720) was amplified by PCRusing a human F11 receptor cDNA as a template (Sobocka et al. (1997)Blood, 90(10): Supp. 1, Part 2, 2996a, incorporated herein by reference)utilizing the forward primer [GCGGGATCCATCGCGATGGGGACAAAGGCG (SEQ ID NO:8)], and the reverse primer [CCGACCTCGAGCGGCATTCCGCTCCACAGCTTCCAT (SEQID NO: 9)] (bases in bold represent BamHI and Xhol sites), respectively.This PCR fragment encodes amino acids ser-1 to asn-208 of F11R, andexcludes the C-terminal transmembrane and cytoplasmic domains. The726-base-pair PCR-product was subcloned into plasmid pcDNA3.1/Myc-His(+) C (Invitrogen, Carlsbad, Calif.) using BamHI and Xhol to yieldpcDNA3.1/F11R. Transcription of the F11R in this plasmid is under thecontrol of CMV immediate-early promoter. The construct pcDNA3.1/F11R wasverified by sequencing and fine restriction mapping prior to its use inexpression studies in COS-7 cells.

Recombinant DNA Methods.

E. coli transformation, plasmid DNA isolation, restriction analysis,extraction of DNA from agarose gells and ligation of insert intopcDNA3.1/myc-His(+)C vector were carried out as described (Sambrook etal. Molecular Cloning: A Laboratory Manuel 2^(nd) Ed. Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. 1989, incorporated herein byreference). Plasmids were isolated from E. coli DH5α (Life Technologies,Grand Island, N.Y.) using Qiaprep columns (Qiagen, Valencia, Calif.).DNA restriction fragments were separated by agarose gel eletrophoresisand isolated with the QIAquick Gel extraction kit (Qiagen). PCR wasperformed using the Perkin Elmer Gene Amp 2400PCR System. DNA sequencingwas performed by PCR-cycle sequencing using ABI PRISM Dye TerminatorCycle Sequencing Kit from Perkin Elmer (Foster City, Calif.) and the ABIPrism 377 DNA Squencer. Computer analysis of sequence data was performedwith the Biology WorkBench, release 3.2.

Transfection of COS-7 Cells.

COS-7 cells were grown in DMEM/10% FBS (Cellgro Mediatech, Inc.) and 1%antimycotic (Life Technologies), at 37° C./5% CO₂ Cells (about 50%confluency), plated in a 75 mm flask, were used for transfection one daylater. The plasmid pcDNA3.1/F11R (10 μg) was transfected into cellsusing 30 μl of FuGENE-6 (Roche Diagnostics). Cells were maintained at37° C./5% CO₂ in 7 ml complete medium. Cells were also treated withFuGENE-6 alone as controls. Total RNA was isolated (RNeasy Mini Kit,Qiagen) and used for subsequent RT-PCR. RNA (2 μg) was used for reversetranscription (Omniscript Reverse Transcriptase, Omniscript RT Kit,Qiagen). Half of the reaction mixture was used to amplify F11R in a35-cycle PCR using the specific F11R primers as detailed previously(Sobocka, supra). PCR cycling was as follows: 94° C. for 5 min followedby 35 cycles of 94° C. for 45 sec, 55° C. for 45 sec, 72° C. for 2 minand 94° C. for 2 min. A 726-bp fragment was obtained only frompcDNA3.1/F11R transfected cells. Conditioned media, collected 72 hposttransfection, were pooled and passed twice over aM.Ab.F11-immunoaffinity column. After washing of the affinity column,the bound sF11R was eluted by use of 50 mM diethylamine (pH 11.5),fractions were collected into 1 M Tris-HCl buffer (pH 8.0) andimmediately dialysed against 10 mM Tris-HCl buffer (pH 7.4). The sF11Rsolution was concentrated 5× by Centricon YM-10 (Bedford, Mass.), andstored frozen at −20° C.

Synthesis of F11R-Peptides.

Five F11R peptides (95% pure) were synthesized (New England Peptides,Inc., Fitchburg, Mass.). The sequence of amino acids in these peptidesand their location within the F11R molecule are shown in Table 1. Theirmass was determined by MALDI-TOF DE mass spectrometry.

TABLE 1  Peptide name PEPTIDE SEQUENCE F11R-peptide 1*SVTVHSSEPEVRIPENNPVKLSC1-------------------------------------------23 (SEQ ID NO: 1)F11R-peptide 2 SYEDRVTFLPTGITFKSVTRED55--------------------------------------76 (SEQ ID NO: 2) F11R-peptide 3WKFDQGDTTRLVEYNNKITASY35-----------------------------------------56 (SEQ ID NO: 3)F11R-peptide 4 KSVTREDTGTYTC 70-----------------------82 (SEQ ID NO: 4)F11R-peptide 5 EQDGSPPSEYTWFKD128-------------------------142 (SEQ ID NO: 5) The amino acid numbersrefer to the sequence of the mature platelet cell surface F11 receptorand of the recombinant protein, sF11R, which does not include the leaderpeptide sequence. *In accordance with the present invention, ser-1 isthe first amino acid that follows the 27 amino acid leader peptidesequence of the nascent protein (Sobocka et al. (2000) Blood95:2600-2609).

Platelet Adhesion to an Immobilized Matrix.

An adhesion assay, based on the determination of cell-derived proteinusing Bicinchoninic Acid (BCA) protein assay (Tuszynski et al. (1990)Anal. Biochem 184:189-191, incorporated herein by reference), was usedfor platelet adhesion to immobilized M.Ab.F11. Wells of a 96-well plate(Nunc-Immuno™ Plate, MaxiSorp™ Surface, flat bottomed) were incubatedovernight at 4° C. with 150 ml of a 1 mg/ml solution of M.Ab.F11. Wellswere aspirated, washed, treated with TBS/1% BSA for 1 h at 37° C., andwashed with TBS/0.1 mM MnCl₂/0.1 mM CaCl₂ Isolated platelet suspensions(100 μl) (3×10⁸/ml) were added and plates were incubated at 37° C. for90 min. Total platelet-associated protein was determined by dissolvingthe attached platelets directly with 100 μl BCA. Platelets wereincubated at 37° C. for 2 h, and absorbance (595 nm) determined(Dynatech Laboratories, Chantilly, Va.).

3D-Structure of Human sF11R.

The crystal structure of the external domain of mouse recombinant JAM(Kostrewa et al. (2001) The Embo J. 20:4391-4398, incorporated herein byreference) was used as a template to generate a 3D model of the humanrecombinant sF11R based on the sequence (Sobocka et al. (2000) Blood95:2600-2609, incorporated herein by reference) of the mature humanplatelet F11R (GenBank accession #AF207907).

Example 2 Preparation and Use of Recombinant sF11R

A secreted, recombinant F11R protein (sF11R) was prepared in COS-7 cellswhich contained only the extracellular portion (amino acids ser-1 toasn-208) of the mature F11R molecule.

The transcription of the recombinant sF11R in COS-7 cells was determinedby RT-PCR. A 726-base-pair fragment was detected only in pcDNA3.1/F11Rtransfected cells. (Babinska et al. (2002) Thromb. Haemost 87:712-721,incorporated herein by reference). To determine the expression of sF11Rin COS-7 cells, the conditioned media obtained from transfected cellswere examined by immunoblotting using both a polyclonal anti-F11Rantibody and the monoclonal M.Ab.F11. The sF11R polypeptide was detectedin the F11R COS-7 conditioned media obtained from these two separateF11R secreting clones, COS-7 cells which were treated with only Fugene 6(mock-transfected), or COS-7 cells transfected with a plasmid lackingF11R DNA, did not secrete sF11R. The sF11R protein was engineered tocontain a Histidine tag sequence, and indeed, it was recognized by ananti-His antibody. The use of a control protein (Positope, 53 kD,obtained from InVitrogen) that contains the His tag, confirmed thisidentification. Finally, sF11R was purified from COS-7 cell media usingM.Ab.F11 immunoaffinity chromatography. The purified sF11R wasrecognized by both the platelet stimulatory monoclonal antibody,M.Ab.F11, and by a polyclonal F11R antibody, directed against theN-terminal amino acids ser-1 to cys-23. The results detailed abovedemonstrate that transfected COS-7 cells not only synthesize by alsosecrete sF11R.

Example 3

Inhibitory effects of the soluble extracellular portion of F11R(“sF11R”), and specific peptides derived thereof. The solublepolypeptide sF11R was tested in the platelet aggregation assay todetermine its effect on the rate and extent of platelet aggregation.

We observed that intact sF11R effectively inhibited M.Ab.F11 (2.45μg/ml)-induced platelet aggregation at two concentrations, 1 μg/ml (FIG.1A) and 4 μg/ml (FIG. 1B). Thus, sF11R appears to possess one or moreinherent structural features, i.e., peptide sequences that participatein platelet aggregation and adhesion.

Example 4 Inhibition of the Potentiation of Agonist-Induced PlateletAggregation by F11R-Specific Peptides

The presence of low, non-aggregating (sub-threshold) concentrations ofthe physiological agonist ADP, collagen or thrombin can cause plateletaggregation when added together with non-aggregating concentrations ofM.Ab.F11 (Sobocka et al. (1997); (Sobocka (1998) PhD Thesis, SUNYDownstate Medical School, Brooklyn, N.Y. Jun. 10, 1998, published Sep.15, 1998); (Sobocka et al. 2001, supra, incorporated herein byreference). Such potentiating effects of M.Ab.F11 on agonist-inducedaggregation are depicted in FIG. 2. When sub-threshold concentrations ofADP (0.5 μM) alone (FIG. 2A) or M.Ab.F11 (0.3 μg/ml) (FIG. 2B),respectively, were added separately to platelet suspensions, there wasno aggregation (low flat baseline tracing shown in panels A and B).However, when sub-threshold concentrations of ADP were added togetherwith sub-threshold concentrations of M.Ab.F11, a pronounced aggregationresponse was observed, reflecting the potentiating effect (see “M.Ab.F11plus ADP” tracings in each panel). Similar potentiating effects wereobserved with sub-threshold levels of thrombin or collagen (data notshown). Synthetic peptides according to published sequences of F11R wereprepared.

The domains within F11R that participate in M.Ab.F11-induced plateletaggregation were identified by the method of Naik et al. (1995) Biochem.J. 311:155-162, incorporated herein by reference. See Table 1.

As shown in FIG. 2D, peptide 1 (SEQ ID NO: 1) (approximately 50 μM)completely inhibited the M.Ab.F11 plus ADP-induced platelet aggregation,and aggregation did not ensue when examined even after a 12 h period. Inaddition peptide 4 (SEQ ID NO: 4) (approximately 50 μM), was also ableto completely block the M.Ab.F11 plus ADP-induced platelet aggregation(FIG. 2C). In contrast, peptides 2 (SEQ ID NO: 2), 3 (SEQ ID NO: 3), or5 (SEQ ID NO: 5) derived from the F11R sequence (see Table 1), had noinhibitory effects on the M.Ab.F11 plus ADP-induced platelet aggregationat concentrations from approximately 50-500 μM. (Not shown).

F11R-Peptides Inhibit the Potentiation of Collagen-Induced PlateletAggregation.

The effect of F11R peptides on M.Ab.F11-induced platelet aggregationpotentiated by non-aggregating concentrations of collagen (0.5 μg/ml)was also examined and provided almost identical results to thosedescribed above and illustrated in FIG. 2A-D. Sub-thresholdconcentrations of collagen and of M.Ab.F11, added together, were shownto induce a strong aggregation. The sub-threshold, non-aggregatingconcentrations (determined separately for the platelets of each blooddonor) of collagen and M.Ab.F11 did not induce platelet aggregation whenadded alone. However, the addition of sub-threshold amounts of collagenand M.Ab.F11 triggered a full-blown platelet aggregation. Thepotentiation by M.Ab.F11 of collagen-induced platelet aggregation couldbe completely blocked by addition of either peptide 1 (SEQ ID NO: 1) orpeptide 4 (SEQ ID NO: 4). However, the peptides of SEQ ID NO: 3 and SEQID NO: 5 did not inhibit platelet aggregation.

The addition of sF11R (1 μg/ml) to platelet suspensions completelyblocked the potentiation by M.Ab.F11 of both ADP- and collagen-inducedplatelet aggregation.

Thus, the results of the studies on inhibiting platelet aggregation werethe same in ADP-induced aggregation as well as collagen-inducedaggregation. Namely, that the sequences comprising peptides 1 (SEQ IDNO: 1) and 4 (SEQ ID NO: 4) in the F11R protein are likely to be keyparticipants in platelet aggregation.

Example 5 Two Specific F11R-Peptides Inhibit Platelet Adhesion toM.Ab.F11

The five peptides with sequences shown in Table 1 were tested also fortheir effects on the adhesion of platelets to immobilized M.Ab.F11(applied at a concentration of 150 ng/well). The left bar of FIG. 3(labeled “M.Ab.F11”) demonstrates the control adhesion measured withoutadded peptide. Peptide 1 (SEQ ID NO: 1), added at 50 μM caused about 60%inhibition of the adhesion of platelets to immobilized M.Ab.F11, andwith 500 μM of peptide 1 (SEQ ID NO: 1), about 70% inhibition wasobserved. Peptide 4 (SEQ ID NO: 4), at 50 μM, produced very little (10%)inhibition compared to peptide 1 (SEQ ID NO: 1) at similarconcentrations. However, 500 μM of peptide 4 (SEQ ID NO: 4) producedapproximately 70% inhibition in the adhesion of platelets to M.Ab.F11,similar to that observed with peptide 1 (SEQ ID NO: 1) at the sameconcentration. On the other hand, the addition of 500 μM (or higher) ofcontrol peptides 2, 3 or 5 did not cause significant inhibition ofplatelet adhesion to M.Ab.F11.

Example 6 Fab Fragments of Monoclonal Antibody M.Ab.F11 Inhibit PlateletAggregation Induced by M.Ab.F11

The F11R/JAM-A receptor is constitutively expressed on the surfacemembrane of human platelets. Studies were carried out testing theeffects of Fab fragments prepared from M.Ab.F11, i.e., on theM.Ab.F11-induced platelet aggregation that is triggered by M.Ab.F11binding to F11R. It was determined that these Fab fragments interferedwith the ability of the intact M.Ab.F11 antibody to activate plateletsthrough the constitutively-expressed F11R.

As shown below, platelet aggregation was assayed in the presence ofeither 0.1 μg/ml (FIG. 4 a), or 1.0 μg/ml (FIG. 4 b). At a concentrationof 1.0 μg/ml (panel b), the Fab fragments completely inhibited theinduction of platelet aggregation induced by the intact stimulatorymonoclonal antibody M.Ab.F11 (approximately 1 μg/ml) (FIG. 4 b). Aten-fold decrease in the concentration of Fab fragments (approximately0.1 μg/ml) was far less effective (FIG. 4 a) indicating a dose dependentrelationship between the concentration of Fab fragments and the extentof platelet aggregation. It has also been observed that the sensitivityof platelets to Fab inhibition of platelet aggregation could vary amongdifferent individuals. Interestingly, the more sensitive the plateletpreparation was to M.Ab.F11-induced platelet aggregation, the moresensitive the platelets were to the Fab-mediated inhibition as well. Inother words, a lower concentration of Fab fragments (lower thanapproximately 1 μg/ml) was sufficient to inhibit M.Ab.F11-inducedplatelet aggregation using the more sensitive platelets. In contrast, Fcfragments of M.Ab.F11 did not inhibit platelet aggregation by intactM.Ab.F11. The Fab fragments blocked M.Ab.F11-induced plateletaggregation competitively presumably, by inhibiting the binding ofM.Ab.F11 to the extracellular domain of F11R. These results verifiedthat the preparations of Fab used in these studies have the expectedactivity of inhibiting interactions of M.Ab.F11 with the F11R.

Example 7

Inhibition of platelet adhesion to TNFa-treated endothelial cells by Fabfragments of M.Ab.F11. Under healthy or normal conditions, theF11R/JAM-A receptor is localized at the tight junctions of endothelialcells (ECs) comprising the endothelium. In healthy subjects platelets donot adhere to the endothelium. However, after contacting a healthyendothelium with one or more proinflammatory (i.e., inducing in theendothelium, specifically the ECs, an inflammatory state or aninflammatory condition) cytokines the localization of F11R shifts awayfrom being concentrated in the junctional regions of the endothelium toa more uniform distribution along the cell surfaces of the ECs in theendothelium. This change in surface distribution of F11R may be referredto as the pathophysiological or inflammatory distribution, which isindicative of the endothelial cells being in an inflammatory state.Therefore, under inflammatory conditions, platelets adhere to theendothelium, in part, due to the enhanced accessibility F11R moleculeson the EC surface for platelet binding.

In experiments presented below (FIG. 5), we tested the inhibitoryeffects of Fab fragments on the adhesion of platelets (eithernon-activated or activated) to human aortic endothelial cells (HAECs)after inducing an inflammatory state by contacting the endothelium withone or more cytokines. As shown in FIG. 5 a, the Fab fragments inhibitedthe adhesion of non-activated platelets to TNFa-treated HAEC by about20% (see FIG. 5 a, BAR 2, 100 μg Fab). However, under conditions inwhich both the platelets were activated with the physiological agonistcollagen and the HAEC were stimulated with a single treatment of TNFα,the level of inhibition was surprisingly found to be complete, i.e.,very close to 100% inhibition (see FIG. 5 b, BAR 2, 100 μg Fab).

Example 8 Fab Fragments of M.Ab.F11 Inhibit the Adhesion of Platelets toHAEC that have been Treated with a Combination of Cytokines

The Inhibitory Efficacy of Fab fragments on the ability of platelets toadhere to inflamed endothelial cells was assessed with HAEC monolayersthat have attained an elevated level of stimulation by treating themonolayers with two cytokines, TNF-α and INF-γ, simultaneously. The useof TNF-α together with INF-γ results in the HAECs achieving aparticularly high level, perhaps even near maximal, of an inflammatorystate that is not achieved by treating HAEC monolayers with either TNF-αor INF-γ individually. TNF-α and INF-γ were applied simultaneously tothe HAEC before measuring the adhesion of platelets to EC. Such aninflammatory condition results in the expression of maximal or nearmaximal F11R/JAM-A at the luminal surface of HAEC, which in turn,results in a maximal degree of adhesion of platelets to the highlystimulated and inflamed HAECs.

As shown in FIG. 6 a, a significant inhibition of the adhesion ofnon-activated platelets to TNF-α plus INF-γ-treated HAEC occurred in thepresence of F11R Fab fragments (BAR 1, no Fab added; BAR 2, 100 μg Fab;BAR 3, 200 μg Fab). These experiments revealed that in contrast to the20% inhibition of the aggregation of non-activated platelets to HAECtreated with the cytokine TNF-α alone, a much greater inhibition(>approximately 60%) (see FIG. 6 a, BAR 2) in the adhesion ofnon-activated platelets was achieved under conditions in which the ECswere exposed to a combined treatment of TNF-α and INF-γ. An even greaterdegree of inhibition (>approximately 80%), in the adhesion ofnon-activated platelets to TNF-α and INF-γ-treated EC could be achievedwhen the concentration of Fab fragments was increased from approximately100 μg/ml to approximately 200 μg/ml (see FIG. 6 a, BAR 3). As shown inFIG. 6 b, under the conditions wherein platelets were activated bycollagen and the EC were stimulated with both TNF-α and INF-γ, weobserved significant inhibition of the adhesion of collagen-activatedplatelets to cytokine-treated HAEC in the presence of Fab fragments (BAR1, no Fab added; BAR 2, 100 μg Fab added; BAR 3, 200 μg Fab added).These experiments revealed that an even greater degree of inhibitioncould be achieved depending on the degree of cytokine activation ofHAEC. A 70% inhibition of the adhesion of collagen-activated plateletsto TNF-α plus INF-γ-treated HAEC could be achieved in the presence ofapproximately 100 μg Fab fragments (BAR 2), and over 90% inhibition ofthe adhesion of collagen-activated platelets to TNF-α plus INF-γ-treatedHAEC could be achieved in the presence of approximately 200 μg Fabfragments (BAR 3).

These results indicate that Fab fragments of either murine M.Ab.F11 orFabs of a humanized version of M.Ab.F11 can be useful as a newtherapeutic for the prevention and treatment of thrombosis,atherosclerosis, heart attack and stroke.

Example 9

The results of an in-vivo study of the effects of F11R peptide 4D onplaque formation in an animal model of atherosclerosis (apoE knockout/KOmice) are discussed below. The atherosclerosis-prone apoE KO is a strainof mice that has been genetically engineered to develop cardiovasculardisease. This genetic abnormality is manifested by the continuousdevelopment of atherosclerotic plaques from early age to adulthood,accompanied by deteriorating, sickly appearance.

The study was conducted on atherosclerosis prone apoE deficient(knock-out female mice of 6 weeks of age) of a C57BL/6 background. Themice were obtained from the Jackson Laboratory (Bar Harbor, Me.). Sevenanimals were injected daily with peptide 4D. In parallel, controlgroups, consisting of animals injected with water, the vehicle used todeliver peptide 4D, was maintained. All mice were fed a Western-typediet.

With the administration of the peptide 4D to the mice for a 5 monthperiod (See FIG. 7B, “+P”), it was observed that the treatment preventedthe degeneration of the animal's appearance over the same time period inwhich the appearance of the untreated group of animals (FIG. 7A)deteriorated. The loss of hair, itching/scratching and lesions thatdeveloped on the skin of the untreated group (See FIG. 7C, arrow) werenot observed in mice treated with peptide 4D (See FIG. 7D, label “+P”).Skin lesions developed in other untreated mice as shown in FIG. 7E,whereas the mouse treated with peptide 4D demonstrated a healthy coat ofhair (See FIG. 7F, label “+P”).

At pre-determined intervals of 3, 4 and 5 months of treatment, theaortae were dissected. Following the dissection of the aortae of allmice, morphometric analyses of lesion areas was performed on aortic archsections stained with Harris hematoxylin-eosin. The total intimal lesionareas were quantified by taking the average of 6 sections spaced 30 μmapart, beginning at the base of the aortic root of each animal. Imageswere viewed and captured with a microscope (Nikon) equipped with a colorvideo camera (Motic Images Plus 2.0) attached to a computerized imagingsystem with Image-J.

Atherosclerotic lesions in the aortic arch are indicated by the arrowsin FIG. 8. A comparison between the large size of the atheroscleroticplaques that developed in apoE KO mice left untreated for three monthswith animals that were treated with peptide 4d is depicted in FIG. 8A.In FIG. 8A, the arrows point to the three major atheroscleroticlesions/plaques within the aortic arch. FIG. 8B compares the size ofplaques in untreated and treated animals following four months ofinjection with peptide 4D for the treated animals. FIG. 8C compares thesize of plaques in untreated and treated animals following five monthsof injections of peptide 4D for the treated animals. The beneficialeffects of treatment with peptide 4D can be observed from these figures.

En face assays (to visualize the entire aorta) were conducted forcomparison of the size and number of atherosclerotic plaques in treatedand untreated animals using an Oil red 0 staining procedure, as shown inFIG. 9. FIG. 9A illustrates the number and size of plaques which wereobserved in animals that were treated with peptide 4D (designated “P1,P2, P3”) for a 3 month period versus animals that were not treated withpeptide 4D (controls numbered “H1, H2, C1, C2, C3”). FIG. 9B illustratesthe differences in the number and size of plaques in animals following a4-month period of injection with peptide 4D, comparing animals “P4, P5”treated with peptide 4D versus controls “H3, H4, H5”. FIG. 9Cillustrates plaques developed in animals following a 5-month period ofinjections with peptide 4D, comparing treated animals “P6, P7” withcontrol untreated animals “H6, H7, C4.” The quantitation of the resultsof the en face assays is shown in FIG. 9D which illustrates that adecrease in the size of the atherosclerotic plaques were detectablefollowing a 3, 4 and 5 month period of injection with peptide 4D.

The proximal aortae of treated an untreated groups were also assessed byhematoxylin-eosin staining of the proximal aorta (root assay) for thesize of the lesions/plaques and the necrotic core area within theproximal aorta. FIG. 10A illustrates the plaques and the size of thenecrotic core area of animals treated or not treated with peptide 4D fora 3 month period. An illustration of the size of the lesion area isshown in FIG. 10B, significant decrease in the size of the lesion areawas observed between untreated animals and animals treated for 3 monthswith peptide 4D (FIG. 10B, apoE KO+P, *P<0.05). Further, as seen in FIG.10C, a significant decrease in the size of the necrosis core area (NE)within the lesion was observed following the 3 month period of injectionwith peptide 4D. FIG. 4D demonstrates that significant decreases in thesize of the plaque/lesion areas were observed in animals following 3, 4and 5 months of injections with peptide 4D.

The results shown above indicate that the administration of F11R peptide4D to apoE KO mice significantly decreases the rate and extent ofatherosclerotic plaque formation in major arterial blood vessels, asshown in FIGS. 8-10, and diminishes the secondary consequences caused byatherosclerosis in the mice, including deterioration of physicalappearance as can be seen in FIG. 7. The results indicate that syntheticcompounds that mimic the structure and inhibitory functions of F11Rpeptides, such as peptide 4D, can serve as drugs for the prevention andtreatment of atherosclerosis, thromboatherosclerosis, heart attacks,stroke and other human cardiovascular disorders involving inflammatoryprocesses and plaque formation.

What is claimed is:
 1. A compound comprising a peptidomimetic whichinteracts sterically with the binding site of a F11R molecule, whereinthe peptidomimetic has a sequence selected from the group consisting ofdKSVTdREDTGTYTC-CONH₂, KSVTdREDTGTYTC-CONH₂, SVTdREDTGTYTC-CONH₂,VTdREDTGTYTC-CONH₂, TdREDTGTYTC-CONH₂, dREDTGTYTC-CONH₂,REDTGTYTC-CONH₂, EDTGTYTC-CONH₂, DTGTYTC-CONH₂, TGTYTC-CONH₂,GTYTC-CONH₂, dKSVTdREDTGTYTC, dKSVTdREDTGTYT, dKSVTdREDTGTY,dKSVTdREDTGT, dKSVTdREDTG, dKSVTdREDT, dKSVTdRED, dKSVTdRE, dKSVTdR,dKSVTd and dKSVT.
 2. The compound of claim 1, wherein the peptidomimetichas a sequence selected from the group consisting ofVTVHSSEPEVRIPENNPVKLSC, TVHSSEPEVRIPENNPVKLSC, VHSSEPEVRIPENNPVKLSC,HSSEPEVRIPENNPVKLSC, SSEPEVRIPENNPVKLSC, SEPEVRIPENNPVKLSC,EPEVRIPENNPVKLSC, PEVRIPENNPVKLSC, EVRIPENNPVKLSC, VRIPENNPVKLSC,RIPENNPVKLSC, IPENNPVKLSC, PENNPVKLSC, ENNPVKLSC, NNPVKLSC, NPVKLSC,PVKLSC, VKLSC, SVTVHSSEPEVRIPENNPVKLS, SVTVHSSEPEVRIPENNPVKL,SVTVHSSEPEVRIPENNPVK, SVTVHSSEPEVRIPENNPV, SVTVHSSEPEVRIPENNP,SVTVHSSEPEVRIPENN, SVT VHSSEPEVRIPEN, SVTVHSSEPEVRIPE, SVTVHSSEPEVRIP,SVTVHSSEPEVRI, SVTVHSSEPEVR, SVTVHSSEPEV, SVTVHSSEPE, SVTVHSSEP,SVTVHSSE, SVTVHSSE, SVTVHSS, SVTVHS and SVTVH.